Process for producing anticancer agent LL-D45042

ABSTRACT

The disclosure describes the production of anticancer agent LL-D45042, having the structure:  
                 
 
by fermentation, to methods for the recovery and concentration of this anticancer agent from crude solutions, and to processes for the purification of this anticancer agent as well as a new microorganism of the species  Streptomyces hygroscopicus  LL-D45042 and mutants thereof useful in the preparation of this compound.

This application is a divisional of copending U.S. patent applicationSer. No. 10/842,277, filed on May 10, 2004, the entire disclosure ofwhich is hereby incorporated by reference, which claims priority fromprovisional Application No. 60/469,719 filed May 12, 2003.

FIELD OF THE INVENTION

The present invention relates to a process for producing an anticanceragent designated LL-D45042 by fermentation, to methods for its recoveryand concentration from crude solutions and to a process for itspurification. The present invention includes within its scope a novelstrain of streptomyces hygroscopicus designated LL-D45042.

BACKGROUND OF THE INVENTION

Rapamycin a macrocyclic lactone has been shown to have antitumoractivity (U.S. Pat No. 4,885,171).

The biological effects of rapamycin are reviewed in TransplantationReviews, 1992, 6, 39-87. Semisynthetic analogs of Rapamycin aredescribed in WO 95/14023 and

further described in WO 93/16189 are macrocyclic lactones produced bythe cultures Streptomyces hygroscopicus (FERM BP-3688), Actinoplanes sp.(FERM BP-3832) or Streptomyces toyocaensis subsp. humicola (ATCC 39471)to produce macrocyclic lactones of the formula

Additional Rapamycin analogs produced by fermentation means described inthe art include: 7,29-bisdesmethyl-rapamycin (U.S. Pat. No. 5,093,338);16-nor-rapamycin (WO 94/10843); 14-methylene rapamycin and 9-methylenerapamycin (WO 93/11130); 7,42-bis (O-demethyl)rapamycin (WO 94/18208)and a process to produce 7-O-dimethylrapamycin by cultivating ATCC55368.

Microbial desmethylation of FK506/FR900520 by actinoplanes sp. ATCC53771 forming products with immunosuppressive activity is reported byTom S. Chen, et al., The Journal of Antibiotics, 45, 118-123 (1992).

Rapamycin structures formed by microbial manipulations of Actinoplanessp. N902-109 are described by Hiroyuki Nishida, et al., The Journal ofAntibiotics, 48, 657-666 (1995).

The precursor feedings and fermentation of s. hygroscopicus LEK III andthe ability to synthesize rapamycins is reported by Lake II Khaw, etal., Journal of Bacteriology, 180, 809-14 (1998).

New antitumor compounds are continually in demand, for the treatment ofcancer in man and the production of new macrolide anticancer compoundsby fermentation means is an important feature of developing antitumoragents for further studies. Equally important are novel strains ofcultures used in the production processes for preparing these compounds.

BRIEF SUMMARY OF THE INVENTION

The invention relates to the production of anticancer agent LL-D45042,by cultivation, to methods for the recovery and concentration of thisanticancer agent from crude solutions, and to processes for thepurification of this anticancer agent as well as a new microorganismuseful in the preparation of this compound.

The invention includes within its scope the anticancer agent in dilutedform, as crude concentrate and in pure form.

The anticancer agent designated LL-D45042 is formed during thecultivation under controlled conditions of a new strain of Streptomyceshygroscopicus, designated LL-D45042.

Cultivation means aerobic growth of an organism in the presence ofassimilable sources of carbon, nitrogen and inorganic anion and cationsalts.The structure of LL-D45042 is:

The physicochemical characteristics of LL-D45042 are as follows:

-   a) Apparent molecular Formula: C₄₉H₇₇NO₁₂-   b) Molecular Weight: Positive Ion Electrospray MS m/z=893.6 (M+Na)⁺;    Negative Ion Electrospray MS m/z=871.1 (M−H)⁻; High Resolution    Fourier Transform MS m/z=894.534230 (M+Na)⁺-   c) Proton Magnetic Resonance Spectrum: (500 MHz d₆-DMSO): See FIG. 1-   d) Carbon-13 Magnetic Resonance Spectrum: (125 MHz d₆-DMSO): See    FIG. 2-   e) Infrared Absorbance Spectrum (thin film, NaCl plates): See. FIG.    3-   f) Ultraviolet Absorption Spectrum: γ_(max) nm    (acetonitrile/water)=270, 280, 292: See FIG. 4

The anticancer agent LL-D45042 is formed during the cultivation undercontrolled conditions of Streptomyces strain designated LL-D45042.

This microorganism is maintained in the culture collection of WyethResearch, Pearl River, N.Y. 10965, as culture LL-D45042. A viableculture of this microorganism is deposited under the Budapest Treatywith the Patent Culture Collection Laboratory, Northern RegionalResearch Center, U.S. Department of Agriculture, Peoria, Ill. 61604, andadded to its permanent collection. Culture LL-D45042 has been assignedthe NRRL accession number NRRL30642.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Characteristic proton nuclear magnetic resonance (NMR) spectrumof LL-D45042 (500 MHz, d₆-DMSO).

FIG. 2 Characteristic carbon nuclear magnetic resonance (NMR) spectrumof LL-D45042 (125 MHz, d₆-DMSO).

FIG. 3 Characteristic infrared (IR) spectrum of LL-D45042 (thin film,NaCl plates).

FIG. 4 Characteristic ultraviolet (UV) spectrum of LL-D45042(acetonitrile/water).

FIG. 5: Immunoblotting data for inhibition of cellular phosphorylationof p70 S6K1 and 4E-BP1 in LNCap prostate cancer cells by LL-D45042.

DESCRIPTION OF LL-D45042

The 16S rDNA sequence is determined for strain LL-D45042 followingisolation and direct sequencing of the amplified gene. The nucleotidesequence is aligned with the sequences of previously studiedstreptomycetes, and phylogenetic trees are generated by using twoneighbour-joining tree algorithms. The 16S rDNA sequence supportedclassification of the strain in the genus and species of S.hygroscopicus.

Observations are made of the cultural, physiological and morphologicalfeatures of culture LL-D45042 using methods well known in the art. Themacromorphology for culture LL-D45042 is described in Table 1 TABLE 1Cultural characteristics of streptomycete strain LL-D45042 Agar MediumLL-D45042 Yeast-malt (ISP2) G: Rapid and abundant AM: Dark grey yellow(91) SM: Dark yellow (88) SP: None Oatmeal (ISP3) G: Very Abundant AM:light grey (264) SM: light yellow brown (76) SP: None Inorganicsalts-starch G: Very Abundant (ISP4) AM: light grey (264) SM: lightolive brown to slight, yellow, brown (74, 94) SP: NoneGlycerol-Asparagine agar G: Abundant (ISP5) AM: light grey (264) SM:dark, grey, yellow (91) SP: NoneG, growth;AM, aerial mycelium;SM, substrate mycelium;SP, soluble pigmentISCC, National Bureau of Standard Centroid Color Charts, Publication440, Washington, D.C. 1976.

Culture LL-D45042 sporulates moderately to abundantly on most mediastudied. Aerial mycelium is monopodially branched; sporophores areterminated by spore chains in the form of short, narrow, compact andclosed coils (Spiral) of three or more turns with ten or more sporespresent in each spiral. Spore color mass is predominately in theGrey-color series. Large spore masses predominate forming blackhygroscopic patches after absorption of water, giving the appearance ofblack, gelatinous spots on the surface of several solid media.

Physiological studies from culture LL-D45042 resulted in no melaninproduction, slow starch hydrolysis, decomposition and fair growth oncellulose, no hydrogen sulfide production, and good digestion of casein.Carbohydrate utilization tests indicated good growth on: D-glucose,L-arabinose, sucrose, I-inositol, D-mannitol, β-D-fructose, α-Lrhammnose, moderate growth on: D-xylose, and cellulose. CultureLL-D45042 exhibited abundant growth at 22° C., 28° C., and 37° C., butno growth at 45° C. and 50° C.

On the basis of the above properties, culture LL-D45042 is most similarto the properties of Streptomyces hygroscopicus. AYB-994 and S.hygroscopicus FERM BP-3688. A comparison of culture LL-D45042 to thesetwo strains of S. hygroscopicus revealed several differentcharacteristics. The cultural characteristics of culture LL-D45042 onglycerol-asparagine agar (ISP5) are abundant growth and dark, grey,yellow surface mycelium in contrast to the poor to moderate growth andcream to pale grey substrate mycelium on the comparison cultures. Inaddition culture LL-D45042 did not produce any soluble pigment on any ofthe ISP agars tested compared to a yellow to cream pigment produced byS. hygroscopicus AY B-994 and S. hygroscopicus FERM BP-3688. ISP carbonutilization tests are also different among the cultures. S.hygroscopicus FERM BP-3688 cannot utilize rhammnose and S. hygroscopicusAY B-994 cannot utilize sucrose or cellulose. Other differences includeS. hygroscopicus FERM BP-3688 produced hydrogen sulfide, the other twocultures do not and culture LL-D45042 can decompose cellulose while theother S. hygroscopicus strains do not. These differences support thecreation of a new strain of S. hygroscopicus designated LL-D45042.

For the production of this anticancer agent LL-D45042 the presentinvention is not limited to this particular organism. In fact, it isdesired and intended to include the use of naturally-occurring mutantsof this organism, as well as induced mutants produced from this organismby various mutagenic means known to those skilled in the art, such asexposure to nitrogen mustard, X-ray radiation, ultraviolet radiation,N′-methyl-N′-nitro-N-nitrosoguanidine, actinophages and the like. It isalso desired and intended to include inter- and intraspecific geneticrecombinants produced by genetic techniques know to those skilled in theart such as for example, conjugation, transduction and geneticengineering techniques.

It is a further embodiment of the invention to provide a method oftreating cancer in mammals in need thereof with an effective amount ofLL-D45042 and pharmaceutically acceptable salts thereof.

It is an additional embodiment of the invention to provide apharmaceutical composition of LL-D45042 and pharmaceutically acceptablesalts thereof in the presence of one or more pharmaceutically acceptablecarriers.

Biological Activity Standard Pharmacological Test Procedures Methods forin vitro Anticancer Test Procedure

The in vitro anticancer activity of LL-D45042 is determined by measuringthe concentration of compound required to inhibit the growth of 50% ofcells in two tumour cell lines. Among the two tumor lines, it waspreviously established that the brain cancer line U87MG is highlysensitive to growth inhibition by rapamycin. The elevated sensitivity isdue to its genetic mutation in the PTEN tumor suppressor gene leading toconstitutive activation of mTOR signaling pathway. In contrast, thebreast cancer line MDA435 does not harbor the PTEN mutation and exhibitsa rapamycin-resistant phenotype. LL-D45042 is also evaluated in abinding assay of FKBP12, a cellular receptor of rapamycin, to determineits binding affinity. The results are given in Table 2, with IC₅₀'sreported in units of ng/mL. TABLE 2 Antiproliferative and FKBP12 bindingActivity for LL-D45042 Growth Inhibition in Protein Binding Cells(ng/mL) (ng/mL) LL-D45042 U87MG MDA435 FKBP 35 >1000 350

These determinations are made by multi-dose testing of LL-D45042 anddirect comparisons to the reference samples.

The inhibitors are further evaluated in the cellular phosphorylationassays by immunoblotting experiments for inhibition of mTOR substratephosphorylation in LNCap prostate cancer cell model (see FIG. 5).

LL-D45042 derives its utility from its anticancer activity. Intherapeutic use, the compound of this invention may be administered inthe form of conventional pharmaceutical compositions appropriate for theintended use. Such compositions may be formulated as to be suitable fororal, parenteral or topical administration. The active ingredient may becombined in admixture with a nontoxic pharmaceutical carrier that maytake a variety of forms depending on the form of preparation desired foradministration, i.e. Oral, parenteral, or topical.

In therapeutic use, the compound of this invention may be administeredin the form of conventional pharmaceutical composition appropriate forthe intended use as an antibacterial. Such compositions may beformulated so as to be suitable for oral, parenteral or topicaladministration. The active ingredient may be combined in admixture withnontoxic pharmaceutical carrier may take a variety of forms, dependingon the form of preparation desired for administration, i.e. oral,parenteral, or topical.

When the compound is employed as an anticancer agent, it may be combinedwith one or more pharmaceutically acceptable carriers, for example,solvents, diluents and the like, and may be administered orally in suchforms as tablets, capsules, dispersible powders, granules, orsuspensions containing, for example, from about 0.05 to 5% of suspendingagent, syrups containing, for example, from about 10 to 50% of sugar,and elixirs containing for example, from about 20 to 50% ethanol and thelike, or parenterally in the form of sterile injectable solutions orsuspensions containing from about 0.05 to 5% suspending agent in anisotonic medium. Such pharmaceutical preparations may contain, forexample, from about 25 to about 90% of the active ingredient incombination with the carrier, more usually between about 5% and 60% byweight.

An effective amount of compound from about 1 mg/kg of body weight toabout 100 mg/kg of body weight should be administered one to five timesper day via any typical route of administration including but notlimited to oral, parenteral (including subcutaneous, intravenous,intramuscular, intrasternal injection or infusion techniques), topicalor rectal, in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. It will beunderstood, however, that the specific dose level and frequency ofdosage for any particular patient may be varied and will depend upon avariety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the age, body weight, general health, sex, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition of the host undergoing therapy.

Additionally, the anticancer effective amount of LL-D45042 of theinvention may be administered at a dosage and frequency without inducingside effects commonly experienced with conventional anticancer therapywhich could include hypersensitivity, neuromuscular blockade, vertigo,photosensitivity, discoloration of teeth, hematologic changes,gastrointestinal disturbances, ototoxicity, and renal, hepatic, orcardiac impairment. Further the frequency and duration of dosage may bemonitored to substantially limit harmful effects to normal tissuescaused by administration at or above the anticancer effective amount ofthe LL-D45042 of the invention.

The active compound may be administered orally as well as byintravenous, intramuscular, or subcutaneous routes. Solid carriersinclude starch, lactose, dicalcium phosphate, microcrystallinecellulose, sucrose and kaolin, while liquid carriers include sterilewater, polyethylene glycols, non-ionic surfactants and edible oils suchas corn, peanut and sesame oils, as are appropriate to the nature of theactive ingredient and the particular form of administration desired.Adjuvants customarily employed in the preparation of pharmaceuticalcompositions may be advantageously included, such as flavoring agents,coloring agents, preserving agents, and antioxidants, for example,vitamin E, ascorbic acid, BHT and BHA. The active compound may also beadministered parenterally or intraperitoneally.

Solutions or suspensions of the active compound as a free base orpharmacologically acceptable salt can be prepared in glycerol, liquid,polyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain apreservative. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacterial and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oil.

The invention accordingly provides a pharmaceutical composition whichcomprises a compound of this invention in combination or associationwith a pharmaceutically acceptable carrier. In particular, the presentinvention provides a pharmaceutical composition which comprises ananticancer effective amount of a compound of this invention and apharmaceutically acceptable carrier.

The present invention further provides a method of treating cancer inwarm-blooded animals including man, which comprises administering to theafflicted warm-blooded animals an anticancer effective amount of acompound or a pharmaceutical composition of a compound of the invention.The invention will be more fully described in conjunction with thefollowing specific examples which are not to be construed as limitingthe scope of the invention.

General Fermentation Conditions

Cultivation of actinomycete designated D45042 may be carried out in awide variety of liquid culture media. Media which are useful for theproduction of hygroscopene include an assimilable source of carbon, suchas dextrin, sucrose, molasses, glycerol, etc.; an assimilable source ofnitrogen, such as protein, protein hydrolysate, polypeptides, aminoacids, corn steep liquor, etc.; and inorganic anions and cations, suchas potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate,chloride, etc. Trace elements such as boron, molybdenum, copper, etc.,are supplied as impurities of other constituents of the media. Aerationin tanks and bottles is supplied by forcing sterile air through or ontothe surface of the fermenting medium.

Further agitation in tanks is provided by a mechanical impeller. Anantifoam agent such as silicon oil may be added as needed.

General Procedure for Preparation of LL-D45042

The cells from a 1 L culture of D45042 are pelleted by centrifugationand exhaustively extracted with 3×200 mL methanol over three days. Themethanol extracts are combined, reduced in vacuo to yield an aqueoussuspension that is subsequently taken to 200 mL total volume 80:20water:methanol. This aqueous solution is extracted with three 150 mLportions of chloroform that are combined, dried over sodium sulfate,filtered, and reduced in vacuo to yield an orange gummy residue. Thismaterial is chromatographed on reversed phase silica (column: 250×20 mmYMC ODS-A RP) employing a gradient of from 30% methanol in water to 95%methanol in 60 minutes. The fraction containing crude LL-D45042 elutedwith approximately 75% methanol (t_(R)=32 minutes). This fraction isfurther purified via reversed phase chromatography (column: 250×10 mmYMC ODS-A RP) employing a gradient from 5% acetonitrile in water to 95%acetonitrile in 20 minutes. Repeated fractionation of all the materialobtained from methanolic extracts of mycelia from 1 L of D45042 yieldedapproximately 0.5 mg of a white solid, (t_(R)=28.9 min). HRFTESIMS:calcd for C₄₉H₇₇NO₁₂Na: 894.533799 found: 894.534230 (−0.00043); UV(acetonitrile-water): 270, 280, 292 nm; ¹H NMR (CD₃CN, 500 MHz) see FIG.1.

1. A process for the preparation of LL-D45042 which comprisescultivating Streptomyces hygroscopicus LL-D45042 or a mutant thereofunder aerobic conditions, in a sterile liquid medium containingassimilable sources of carbon, nitrogen and inorganic anion and cationsalts, until LL-D45042 is formed and recovering and isolating LL-D45042.2. A biologically pure culture Streptomyces hygroscopicus LL-D45042 or aLL-D45042 producing mutant thereof.
 3. A biologically pure culture ofthe microorganism Streptomyces hygroscopicus, LL-D45042 or a mutantthereof, having the ability to produce a compound having the structure

on aerobic fermentation in a nutrient medium.
 4. A method of treating amammal affected by cancer, which method comprises administering to saidmammal an effective amount of a compound having the structure.


5. The compound having the structure